Oligonucleotides have recently become accepted as therapeutic moieties in the treatment of disease states in animals and man. For example, workers in the field have now identified antisense, triplex, mimetic or "decoy" and other oligonucleotide therapeutic compositions which are capable of modulating expression of genes implicated in vital, fungal and metabolic diseases.
U.S. Pat. No. 5,098,890 is directed to antisense oligonucleotide therapies for certain cancerous conditions. U.S. Pat. No. 5,135,917 provides antisense oligonucleotides that inhibit human interleukin-1 receptor expression. U.S. Pat. No. 5,087,617 provides methods for treating cancer patients with antisense oligonucleotides. U.S. Pat. No. 5,166,195 provides oligonucleotide inhibitors of HIV. U.S. Pat. No. 5,004,810 provides oligomers capable of hybridizing to herpes simplex virus Vmw65 mRNA and inhibiting replication. U.S. Pat. No. 5,194,428 provides antisense oligonucleotides having antiviral activity against influenzavirus. U.S. Pat. No. 4,806,463 provides antisense oligonucleotides and methods of using them to inhibit HTLV-III replication. U.S. Pat. No. 5,157,115 provides nucleic acid compositions which inhibit or control IL-2 genes by competitively binding to their transcription factors. U.S. Pat. No. 5,176,996 teaches methods for making synthetic oligonucleotides which bind tc target sequences in a duplex DNA forming collinear triplexes. Oligonucleotides have been safely administered to humans and several clinical trials are presently underway. It is, thus, established that oligonucleotides can be useful therapeutic instrumentalities and can be configured to be useful in treatment regimes for treatment of cells and animals, especially humans.
One method for inhibiting specific gene expression using oligonucleotides is the antisense approach, in which oligonucleotides are designed to be complementary to a specific target messenger RNA (mRNA), thereby modulating the activity of the mRNA. "Hybridization," in the context of this invention, means hydrogen bonding, also known as Watson-Crick base pairing, between complementary bases, usually on opposite nucleic acid strands or two regions of a nucleic acid strand. Guanine and cytosine are examples of complementary bases which are known to form three hydrogen bonds between them. Adenine and thymine are examples of complementary bases which are known to form two hydrogen bonds between them. "Specifically hybridizable" indicates a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences. It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable.
The relationship between an oligonucleotide and its complementary target nucleic acid is commonly denoted as "antisense." Recent reviews of the antisense field include Uhlmann, E. and A. Peyman (1990) Chem. Reviews 90:544-584; Dolnick, B. J., (1991) Cancer Invest. 9: 185-194; and Stein, C. A. and Y.-C. Cheng (1993) Science 261:1004-1012.
Another approach to oligonucleotide therapeutics is the triplexing (triple-strand) approach, in which oligonucleotides are designed to bind to double-stranded DNA targets, forming triple-stranded structures. Triple helix-forming oligonucleotides are expected to inhibit transcription of the target gene either by inhibition of transcription factor binding or by directly blocking transcription, thereby modulating the expression of the gene. Grigoriev et al., (1993) Proc. Natl. Acad. Sci. U.S.A., 90: 3501-3505.
Oligonucleotides can also be used as "decoys" or mimetics of native DNA or RNA in order to compete with native sequences for binding of specific DNA- or RNA-binding proteins. For example, U.S. Pat. No. 5,157,115 provides nucleic acid sequences which competitively bind regions of the IL-2 or IL-2.alpha. genes corresponding to their respective transcription factors. Bielinska et al. disclose inhibition of sequence-specific transcription factor binding with double-stranded oligonucleotides containing known consensus sequences for transcription factor binding. Science 250:997-1000 (1990).
The above approaches are dependent upon a known target sequence, for which a complementary or mimetic oligonucleotide sequence is designed. This approach can be described as a rational drug design approach. Strategies have also been developed for in vitro screening of large populations of random oligonucleotides, by which oligonucleotides having a desired activity such as activity against a preselected target molecule can be identified from a random pool. An advantage of these strategies is that they enable active oligonucleotides to be found whose activity may not have been predicted based purely on the oligonucleotide sequence. This may be because, for example, a target molecule was not known or was not appreciated as a good antisense target, because the active oligonucleotide binds to a region of target secondary structure rather than a linear sequence, or because the "target molecule" is a complex rather than a single molecule. Another advantage is that the target molecule is not limited to nucleic acid, and in fact does not have to be identified at all, as long as a desired activity can be assayed. The desired activity can be, for example, mimetic, catalytic or enzymatic activity in addition to binding activity. Binding is not limited to base-pairing to a complementary target molecule.
Examples of such in vitro random oligonucleotide screening strategies are disclosed in PCT publications WO 93/05182, which discloses an iterative method of determining an oligonucleotide having specific activity for a target biomolecule, and WO 93/04204, which teaches a method for determining an oligomer having specific activity for a target molecule from a pool of random subunits by repeated syntheses of increasingly defined oligomers coupled with selection procedures. PCT publication WO 92/00091 discloses a library of bio-oligomers attached to solid phase supports and use of the library to determine the sequence of a bio-oligomer ligand for an acceptor molecule. PCT publication WO 91/19813 teaches a method for identifying nucleic acid ligands from a mixture of nucleic acids by iterative binding and separation. PCT publication WO 92/14842 discloses isolation of single-stranded DNA oligonucleotide "aptamers" which bind thrombin and inhibit its function in vitro. While these strategies have proven useful in identifying desirable oligonucleotide compounds in vitro, the activity of an oligonucleotide compound measured in vitro may differ from the activity of the same compound in the intracellular microenvironment. It is therefore desirable to screen random oligonucleotides for in vivo activity.
PCT publication WO 86/05803 discloses use of genes at least partially composed of stochastic synthetic polynucleotides and introduced into host cells, and identification of peptides or polypeptides produced, or DNA or RNA sequences incorporated into the host cell genome, which have a desired property. PCT publication W0 92/07071 discloses a method for obtaining genetic suppressor elements for a known gene comprising fragmentation of a DNA sequence homologous to the gene to be suppressed, incorporation of the DNA fragments into vectors, introduction of the vectors into cells, and isolation of genetically modified cells containing genetic suppressor elements.
There has been and continues to be a long-felt need for oligonucleotides which are capable of effective therapeutic and diagnostic use. Further, there is a need for a method of identifying oligonucleotides which have activity in vivo particularly when a target sequence or structure is not defined.